The blood coagulation enzyme thrombin is being investigated in an attempt to understand better its restrictive specificity for small molecule and oligopeptide substrates when compared to that of closely related proteases, such as trypsin and plasmin. Human thrombin is used because of its medical importance and its availability from Cohn fraction III as a by-product of commercial fractionation of human blood. Studies in progress include (1) the improvement of methodology for preparing, stabilizing, and evaluating highly purified thrombin; (2) the synthesis and testing of potential small molecule substrates and inhibitors for thrombin; (3) the synthesis and testing of potential oligopeptide substrates for thrombin to determine optimal binding requirements; and (4) the design and examination of affinity-labeling reagents for labeling portions of the active site of thrombin which direct or restrict its binding specificities. These studies have contributed simplified procedures for high yields of thrombin with high specific activities and have already shown that thrombin specificity resides both adjacent to and removed from the catalytic portion of the active site. Further studies will be extended to the amino acid sequencing of limited active site peptides identified by affinity- labeling reagents in an attempt to correlate them with the known sequence of bovine thrombin.